Signal enhancement system with multiple labeled-moieties

ABSTRACT

Dipstick tests for detecting analyte are described. In a preferred embodiment, a multiple biotinylated antibody capable of binding analyte is bound to an anti-biotin antibody labeled with colloidal gold and wicked up the dipstick with test solution thought to contain analyte. Complex formed between analyte, biotinylated anti-analyte antibody, and colloidal gold labeled anti-biotin antibody is captured at a capture zone of the dipstick. Presence of colloidal gold label at the capture zone indicates the presence of analyte in the test solution. The sensitivity of analyte detection using such methods is an order of magnitude higher than for comparable methods in which biotinylated anti-analyte antibody bound to analyte is wicked up the dipstick in a first step, and a colloidal gold labeled anti-biotin antibody is wicked up the dipstick in a separate step. Kits for performing the tests of the invention are also described.

This invention relates to methods for testing for the presence of ananalyte in a test solution and to kits for carrying out such methods.

One conventional method for testing for the presence of an analyte in atest solution comprises capturing the analyte on a dipstick anddetecting for the presence of the analyte on the dipstick. The dipstickhas a contact end for contacting the test solution and a capture zoneremote from the contact end to which an anti-analyte antibody (thecapture antibody) is immobilized.

To test for the presence of analyte, the contact end of the dipstick iscontacted with the test solution. If analyte is present in the testsolution it travels to the capture zone of the dipstick by capillaryaction where it is captured by the capture antibody. The presence ofanalyte at the capture zone of the dipstick is detected by a furtheranti-analyte antibody (the detection antibody) labelled with, forexample, colloidal gold.

These dipstick tests have several advantages. They are easy and cheap toperform, no specialist instruments are required, and the results areobtained rapidly and can be read visually. These tests are, therefore,particularly suited for use in a physician's office, at home, in remoteareas, and in developing countries where specialist equipment may not beavailable. They can be used, for example, to test whether a patient isinfected with a disease causing micro-organism such as Chlamydiatrachomatis.

However, the sensitivity of analyte detection using such tests isrelatively low. Consequently, if the analyte is only present in smallamounts in the test solution it can remain undetected. This is aparticular disadvantage if the test is being used to diagnose whether ornot a patient has a particular disease as the patient may not bediagnosed as having that disease. This is particularly the case wherethe patient is asymptomatic, but also where symptoms are present but itis necessary to confirm that these are caused by a particular disease,or particular disease strain.

WO 00/25135 discloses a two-step dipstick detection system for detectingantigen. The detection system uses an anti-antigen biotinylated antibodyand an anti-biotin antibody labelled with colloidal gold (anti-biotingold conjugate). In the first step, the biotinylated antibody is mixedwith the test solution and the tip of the dipstick membrane is thenimmersed in the mixture so that the aqueous mixture wicks up themembrane by capillary action. Antigen in the mixture bound to thebiotinylated antibody is captured by an immobilised anti-antigenantibody at a capture zone of the dipstick to form a complex comprisingthe immobilised anti-antigen antibody, the antigen and the biotinylatedantibody. In the second step, the dipstick is immersed in a separatesuspension of anti-biotin gold conjugate. The anti-biotin gold conjugatewicks up the membrane by capillary action and binds to biotinylatedantibody captured with the antigen at the capture zone. Antigen is thendetected by the presence of gold label at the capture zone.

Whilst the two-step system may provide increased sensitivity, it isdesired to further improve the sensitivity of analyte detection.

According to a first aspect of the invention there is provided a methodfor testing for the presence of an analyte in a test solution whichcomprises the following steps:

a) providing a chromatographic strip having a contact end for contactingthe test solution and a capture moiety immobilised at a capture zone ofthe chromatographic strip remote from the contact end, the capturemoiety comprising a member of a ligand/anti-ligand binding pair capableof binding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair;b) contacting a targeting agent, capable of binding the analyte orderivative thereof, with the test solution to allow binding of thetargeting agent to analyte or derivative in the test solution, thetargeting agent being provided with a plurality of ligands;c) binding a label to each of two or more ligands of the is targetingagent;d) contacting the contact end of the chromatographic strip with the testsolution to allow analyte, or derivative thereof, bound to the labelledtargeting agent to travel to the capture zone by capillary action and becaptured by the capture moiety; ande) detecting for the presence of label at the capture zone.

According to the invention the targeting agent is bound to the labels toform the labelled targeting agent before it contacts the capture zone.This is in contrast to the method of WO 00/25135 in which thebiotinylated antibody and the anti-biotin gold conjugate are wicked upthe dipstick membrane in two separate steps. The sensitivity of analytedetection using methods of the invention has surprisingly been found tobe greater than the two-step method of WO 00/25135. Under optimalconditions, the sensitivity of analyte detection is at least an order ofmagnitude higher than the sensitivity of detection using the two-stepmethod.

To perform a method of the first aspect of the invention, the targetingagent and labels may simply be added to the test solution and the testsolution then contacted with the contact end of the chromatographicstrip. Such methods are easier to perform than the method disclosed inWO 00/25135 in which two separate wicking steps are required. Theresults may, therefore, be obtained more rapidly, and yet thesensitivity of analyte detection is higher.

The term “chromatographic strip” is used herein to mean any porous stripof material capable of transporting a solution by capillarity. Thechromatographic strip may be capable of bibulous or non bibulous lateralflow, but preferably bibulous lateral flow. By the term “non-bibulouslateral flow” is meant liquid flow in which all of the dissolved ordispersed components of the liquid are carried at substantially equalrates and with relatively unimpaired flow laterally through the membraneas opposed to preferential retention of one or more components as wouldoccur with “bibulous lateral flow”. Materials capable of bibulouslateral flow include paper, nitrocellulose, and nylon. A preferredexample is nitrocellulose.

The labels may be bound to the ligands of the targeting agent bypre-mixing the targeting agent with the labels before the targetingagent is added to (or otherwise contacted with) the test solution.However, in some circumstances, it is preferred that the targeting agentand labels are not pre-mixed because such pre-mixing can cause thetargeting agent and labels to precipitate. Thus, the targeting agent andthe labels may be added separately to (or contacted separately with) thetest solution. The targeting agent and the labels can be added to (orcontacted with) the test solution at substantially the same time, or inany order.

The test solution may be pre-incubated with the targeting agent andlabels before the test solution is contacted with the contact end of thechromatographic strip to ensure complex formation. The optimal time ofpre-incubation will depend on the ratio of the reagents and the flowrate of the chromatographic strip. In some cases, pre-incubation for toolong can decrease the detection signal obtained, and even lead to falsepositive detection signals. Thus, it may be necessary to optimise thepre-incubation time for the particular conditions used.

It may be desired to pre-incubate the targeting agent with the testsolution before binding the labels to the targeting agent so that thetargeting agent can be allowed to bind to analyte in the test solutionunder optimum binding conditions.

In alternative aspects of the invention, binding of the labels to thetargeting agent may take place on the chromatographic strip. This can beachieved by releasably immobilising the labels and/or targeting agent tothe chromatographic strip at a conjugate zone between the contact endand the capture zone. The labels and/or targeting agent are thenreleased into test solution as it travels by capillary action to thecapture zone. If the labels, but not the targeting agent, are releasablyimmobilised, the targeting agent should be contacted with the testsolution so that targeting agent in the test solution can bind to thelabels as they are releasably immobilised. Similarly, if the targetingagent is releasably immobilised, but not the labels, the labels shouldbe contacted with the test solution.

Thus, according to a second aspect of the invention there is provided amethod for testing for the presence of an analyte in a test solutionwhich comprises the following steps:

a) providing a chromatographic strip having:i) a contact end for contacting the test solution;ii) a capture moiety immobilised at a capture zone of thechromatographic strip remote from the contact end, the capture moietycomprising a member of a ligand/anti-ligand binding pair capable ofbinding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair; andiii) a targeting agent releasably immobilised at a conjugate zone of thechromatographic strip between the contact end and the capture zone, thetargeting agent being provided with a plurality of ligands and beingcapable of binding the analyte or derivative thereof;b) contacting a plurality of labels with the test solution;c) contacting the contact end of the chromatographic strip with the testsolution to allow test solution to travel is through the conjugate zoneto the capture zone, thereby releasing targeting agent from theconjugate zone so that each of two or more ligands of the releasedtargeting agent are bound by a label and so that released targetingagent bound to the labels can travel with analyte or derivative in thetest solution to the capture zone and be captured at the capture zone aspart of a complex formed between the capture moiety, analyte orderivative, the targeting agent, and the labels; andd) detecting for the presence of label at the capture zone.

Alternatively, the labels may be releasably immobilised to the conjugatezone of the chromatographic strip, and the targeting agent contactedwith the test solution, or both the labels and the targeting agent maybe releasably immobilised to the conjugate zone.

Immobilisation may be carried out simply by drying the labels and/ortargeting agent onto the chromatographic strip. An advantage ofreleasably immobilising some or all of the reagents necessary to performa method of the invention onto the chromatographic strip is that thesereagents do not then need to be added separately to the test solution,nor carried or packaged separately with the other components necessaryto perform the method.

In other aspects of the invention, it may be desirable to contact thecontact end of the chromatographic strip with a solution containing thelabelled targeting agent after the contact end has been contacted withthe test solution, or to contact the targeting agent and labels with thetest solution after analyte or derivative has been allowed to travel bycapillary action to the capture zone, so that labelled targeting agenttravels by capillary action to the capture zone separately from theanalyte or derivative.

A possible advantage of such aspects is that any non specific binding oflabels to the chromatographic strip can is be reduced because more ofthe test solution (or solution containing the targeting agent) travelsthrough the chromatographic strip, thereby washing the chromatographicstrip.

In other aspects of the invention, the labelled targeting agent may becontacted directly with the capture zone of the chromatographic stripafter analyte or derivative has been captured by the capture moiety.Binding of the labels to the ligands of the targeting agent should becarried out before (or, exceptionally, as) the targeting agent iscontacted with the capture zone.

Thus, in its broadest sense a method of the invention for testing forthe presence of an analyte in a test solution comprises the followingsteps:

a) providing a chromatographic strip having a contact end for contactingthe test solution and a capture moiety immobilised at a capture zone ofthe chromatographic strip, the capture moiety comprising a member of aligand/anti-ligand binding pair capable (other than by nucleic acid basepairing interaction) of binding the analyte, or a derivative thereof, asthe other member of the ligand/anti-ligand binding pair;b) contacting the contact end of the chromatographic strip with the testsolution to allow analyte, or derivative thereof, to travel to thecapture zone by capillary action and be captured by the capture moiety;c) contacting a labelled targeting agent with the capture zone, thelabelled targeting agent being capable of binding the analyte, or aderivative thereof, thereby allowing labelled targeting agent to becaptured at the capture zone as part of a complex comprising the capturemoiety, the analyte or derivative and the labelled targeting agent,wherein the labelled targeting agent comprises a plurality of ligandsand each of two or more ligands are bound to a label; andd) detecting for the presence of label at the capture zone.

In some aspects and embodiments of the invention, it may be desirablefor the labels and/or the targeting agent to be in dry form, for exampleas a powder or tablet which is contacted with the test solution. Thishas the advantage that kits for performing methods of the invention canbe reduced in weight and size because they do not need to includeseparate solutions of the labels and/or targeting agent. This adds tothe ease of use of kits of the invention, and can be important if thekits are to be transported, particularly to remote areas. A furtheradvantage is that the targeting agent and/or label may be more stable indry form. This may be particularly important when methods of theinvention are performed in areas where it may be difficult to freeze orcool solutions of reagents which would otherwise be unstable at roomtemperature.

Preferably the targeting agent has at least 4 ligands, more preferablyat least 6 ligands. Preferably the targeting agent has no more than 50ligands. The optimum number of ligands may depend on the identity of thetargeting agent. For example, for a targeting agent comprising IgG, thenumber of ligands preferably does not exceed 20 per IgG molecule.However, for a targeting agent comprising IgM, more ligands may be used.

We have found that the optimum number of ligands of the targeting agentdepends on the flow rate of the chromatographic strip. In general, thefaster the flow rate, the higher the number of ligands per targetingagent.

The flow rate of the membrane depends on the pore size, pore structure,and the surfactant treatment of the membrane. In untreatednitrocellulose membranes, flow rates are influenced by pore size: thelarger the pore, the faster the flow rate. However, post treatments andblocking solutions can also have significant impact on the flow rate.Different solutions may also flow along a membrane at different rates,whether by virtue of their viscosity or particulate content.

A fast flow rate membrane is defined here as a membrane in which watertravels by capillary action at a rate of about 70-80 seconds per 40 mm.Examples of fast flow rate membranes are: Whatman Purabind A-RP membrane(pore size 8 μm)−water flow rate=80 seconds per 40 mm; and WhatmanPurabind A-XP membrane (pore size 12 μm)−water flow rate=70 seconds per40 mm. An example of a slower flow rate membrane is Schleicher & SchuellAE99 membrane (pore size 8 μm)−water flow rate 130 seconds per 40 mm.

For fast flow rate membranes, optimum results are obtained with about9-20 ligands per targeting agent, preferably about 14-18. For slowerflow rate membranes optimum results are obtained with about 6-12 ligandsper targeting agent, more preferably 8-12 ligands, even more preferably8-10 ligands, and most preferably 8 or 9 ligands.

We have found that the optimum number of ligands of the targeting agentdepends on the viscosity of the biological sample. In general, the moreviscous the sample type, the lower the number of ligands per targetingagent. For example, where a method of the invention is used to detectCT, typical test samples are urine, endocervical swab, or urethral swab.These samples have different viscosities, so the optimum number ofligands per targeting agent used will depend on the viscosity of thesample solution.

The targeting agent may comprise a single moiety, or more than onemoiety. For example the targeting agent may comprise: a primary moietycapable of binding to the analyte or derivative; and a secondary moietycapable of binding to the primary moiety, the secondary moiety beingprovided with a plurality of ligands.

Preferred ligands include biotin (which can be bound by anti-biotinantibody, avidin, streptavidin, or a derivative thereof), fluorescein(which can be bound by anti-fluorescein antibody) and DNP (which can bebound by anti-DNP antibody).

The or each moiety of the targeting agent is preferably an antibody. Theterm “antibody” as used herein means any antibody or antibody fragment(whether produced naturally or recombinantly) which retains antigenbinding activity. This includes a monoclonal or polyclonal antibody, asingle chain antibody, a Fab fragment of a monoclonal or polyclonalantibody, and a chimeric antibody.

In a preferred embodiment, the targeting agent comprises a primaryantibody capable of binding to the analyte or derivative, and asecondary antibody capable of binding to the primary antibody, thesecondary antibody being covalently coupled to a plurality of ligands.This embodiment is preferred because the secondary antibody can beprepared separately and used with different primary antibodies fordetecting different analytes.

A plurality of labels are preferably bound to at least one of theligands of the targeting agent, thereby further increasing thesensitivity of analyte detection.

Any labels which, when part of a complex formed between the capturemoiety, analyte or derivative, the targeting agent and the labels, allowdetection of the complex on the chromatographic strip may be used.Preferred labels are visually detectable labels. Examples of suitablevisually detectable labels include textile dyes and coloured particlessuch as coloured latex particles and metal sol such as colloidal gold orselenium. Colloidal gold with a size range of about 20-60 nm ispreferred, more preferably 20-40 nm. Similar assay sensitivities can beobtained using colloidal gold with a narrow size distribution, from23-31 nm (30 nm British Biocell International Limited), or a wide sizedistribution, from 20-47 nm (average 29-38 nm).

Examples of other suitable labels include radioactive labels,luminescent labels particularly fluorescent labels, and labelscomprising an enzyme capable of reacting with a chromogenic substrate orwith a substrate which is converted into a luminescent product by theenzyme. Such chemiluminescent labels are particularly preferred becausethe strength of the luminescent signal generated is high in relation toother labels, thereby enhancing the sensitivity of analyte detection.

Each label may be provided by a labelling agent. Each labelling agentmay comprise a single moiety or more than one moiety. For example, alabelling agent may comprise: a primary moiety capable of binding to aligand of the targeting agent; and a labelled secondary moiety capableof binding to the primary moiety.

The or each moiety of the labelling agent is preferably an antibody.

In a preferred embodiment, the labelling agent comprises a primaryantibody capable of binding to a ligand of the targeting agent and asecondary antibody capable of binding to the primary antibody, thesecondary antibody being coupled to a label. This embodiment ispreferred because the secondary antibody can be prepared separately andused with different primary antibodies for binding to different ligands.

For embodiments in which the ligands of the targeting agent comprisebiotin, suitable labelling agents include the following:

i) a labelled anti-biotin antibody;ii) a labelled moiety comprising avidin, streptavidin, or a derivativethereof which retains biotin binding activity;iii) an anti-biotin antibody, avidin, streptavidin, or a derivativethereof which retains biotin binding activity (the primary moiety) and alabelled antibody (the secondary moiety) capable of binding to theprimary moiety.

Each labelling agent may preferably comprise a plurality of labels tofurther enhance the sensitivity of detection. This may be achieved bycovalently coupling a plurality of labels to each labelling agent.Alternatively, if the labelling agent comprises more than one moiety,each moiety may be labelled, as shown in FIG. 5C.

The capture moiety may comprise a single moiety or a plurality ofmoieties non covalently bound together. The capture moiety may comprisean antibody capable of binding the analyte or derivative thereof.

In other embodiments, the analyte may be an antibody and the capturemoiety an antigen. Such embodiments can be particularly advantageous fortesting whether or not an individual has been infected with a diseasecausing micro-organism if the amount of antigen of that disease causingmicro-organism in the test solution is likely to be very low. Antibodiesproduced by the individual to the antigen in response to infection bythe micro-organism may instead be present at much higher amounts,thereby making detection of the antibodies a more sensitive way ofdiagnosing infection. Examples are antibodies produced in response toHIV infection.

A derivative of the analyte may be formed by chemically modifying theanalyte for example by covalently coupling the analyte to a ligand whichcan be bound by the capture moiety. In one embodiment, the analyte couldbe covalently coupled to biotin to form a derivative of the analytewhich can then be captured by a capture moiety comprising an anti-biotinantibody, avidin, streptavidin, or a biotin binding derivative thereof.Alternatively, the analyte may be formed by non covalently binding aderivatizing moiety, such as an antibody, to the analyte which can bebound by the capture moiety.

An advantage of forming a derivative of the analyte is that the analyteand labelled targeting agent may be further spaced from thechromatographic strip, thus reducing or preventing any steric hindrancewhich might otherwise occur between the chromatographic strip and ananalyte-containing complex captured at the capture zone. Capture of ananalyte derivative also allows identical chromatographic strips to beused to capture different analytes if the analyte derivative comprises acapture ligand which can be bound by the capture moiety of such strips.

In preferred embodiments, the analyte derivative comprises a pluralityof capture ligands, each of which can be bound by the capture moiety.The presence of a plurality of capture ligands increases the probabilitythat the analyte derivative will be captured by the capture moiety.

Examples of preferred embodiments in which the analyte is bound by aderivatizing moiety are shown in FIG. 6. Note that in this figure,binding of analyte by labelled targeting moiety is not shown.

Methods of the invention may be used to test for any suitable analyte.Examples include antigens of infectious agents or antibodies raisedagainst such antigens, hormones is (for example as a pregnancy test),metabolites (for example to test for metabolic disorders), drugs(therapeutic or illicit), vitamins, steroids, or antibodies produced aspart of an allergic reaction.

Preferred examples of antigens of infectious agents or antibodies tosuch antigens are: hepatitis B virus surface antigen (HBsAg), HBsAg ‘a’epitope, hepatitis B ‘e’ antigen (HBeAg), antibodies to HBeAg,antibodies to hepatitis B core antigen (e.g. anti hepatitis B coreantigen-IgG and -IgM), hepatitis C virus (HCV) antigens, antibodies toHCV antigens, HIV antigens (of HIV 1 or HIV 2), antibodies to HIVantigens, Chlamydia trachomatis antigens, or Neisseria gonorrheaantigens.

There are also provided according to the invention kits for performingmethods of the invention.

A kit for performing a method of the first aspect of the inventioncomprises:

i) a chromatographic strip having a contact end for contacting the testsolution and a capture moiety immobilised at a capture zone of thechromatographic strip remote from the contact end, the capture moietycomprising a member of a ligand/anti-ligand binding pair capable ofbinding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair;ii) separately from the chromatographic strip, a targeting agent capableof binding the analyte or derivative thereof, the targeting agent beingprovided with a plurality of ligands, and a plurality of labellingagents each bound to a ligand of the targeting moiety, each labellingagent being provided with a label.

A kit for performing a method of the second aspect of the inventioncomprises:

i) a chromatographic strip having: a contact end for contacting the testsolution; a capture moiety immobilised at a capture zone of thechromatographic strip remote from the contact end, the capture moietycomprising a member of a ligand/anti-ligand binding pair capable ofbinding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair; and a targeting agent releasablyimmobilised at a conjugate zone of the chromatographic strip between thecontact end and the capture zone, the targeting agent being providedwith a plurality of ligands and being capable of binding the analyte orderivative thereof; and separately ii) a plurality of labelling agentscapable of binding to a ligand of the targeting moiety, each labellingagent being provided with a label.

The labelling agents may be releasably immobilised to the conjugate zoneof the chromatographic strip as well as the targeting agent.Alternatively, the labelling agents may be immobilised to thechromatographic strip instead of the targeting agent.

Where the kit is for performing a method of the second aspect of theinvention, it will be appreciated that releasably immobilising thetargeting agent and the labelling agents to the conjugate zone of thechromatographic strip has the particular advantage that all the reagentsnecessary for testing for the presence of the analyte in the testsolution are present on the chromatographic strip (provided thatlabelling agents are chosen which do not require reaction with furtherreagents is to visualize the complex captured at the capture zone,unless these further reagents are also releasably immobilised to thechromatographic strip).

In such embodiments, it will be necessary to ensure that there issufficient distance between the conjugate zone and the capture zone forthe labelling agents to be able to bind to the targeting agent as theytravel to the capture zone once they have been released. Alternatively,the conjugate zone should be immersed in the test solution to allowrelease of the targeting agent and the labelling agents into the testsolution so that they can bind to each other in the test solution beforetravelling by capillary action to the capture zone.

Where the targeting agent and the labelling agents are releasablyimmobilised to the conjugate zone of the chromatographic strip, they maybe immobilised to the same part of the conjugate zone or at differentzones within the conjugate zone. Where the targeting agent and thelabelling agents are immobilised to the same part of the conjugate zone,they may be interspersed with one another at the conjugate zone, or theymay be in different layers.

In one arrangement it may be preferred for the labelling agents to beimmobilised directly to the chromatographic strip at the conjugate zone,thereby forming a first layer, and the targeting agent to be immobilisedto the first layer, thereby forming a second layer on top of the firstlayer. A possible advantage of this arrangement is that the targetingagent would be expected to be released into the test solution before thelabelling agents. This may be of importance to ensure efficient complexformation at the capture zone if it is suspected that the labellingagents could interfere with binding of the targeting agent to analyte orderivative.

Where the targeting agent and/or the labelling agent are not immobilisedto the chromatographic strip, they may preferably be in dry form, forexample as a powder or a tablet.

The sensitivity of analyte detection may depend on the ratio of thetargeting agent to the labelling agent. Consequently, the ratio shouldbe chosen to obtain optimum results.

Where kits of the invention comprise a targeting agent consisting ofmore than one moiety, it may in some circumstances be desirable for oneor more of the moieties to be releasably immobilised to thechromatographic strip and one or more of the remaining moieties to beseparate from the chromatographic strip. Similarly, where each labellingagent comprises one or more moieties, it may in some circumstances bedesirable for one or more of the moieties to be releasably immobilisedto the chromatographic strip and one or more of the remaining moietiesto be separate from the chromatographic strip.

Kits of the invention in which a further reagent(s) is required fordetection of a captured complex containing the labelling agent (forexample, those kits in which the labelling agents comprise an enzymecapable of converting a chromogenic substrate or a substrate which isconverted to a luminescent product by the enzyme) preferably furthercomprise the reagent(s) required for detection.

For those aspects and embodiments of the invention in which thetargeting agent and/or the labels are releasably immobilised to aconjugate zone of the chromatographic strip, the concentration of thelabels and/or the targeting agent in the test solution arriving at thecapture zone by capillary action is likely to vary. This is because theconcentration of the targeting agent and/or labels in the is testsolution will rise and fall as the releasably immobilised targetingagent and/or labelling agent is released into the test solution untilthere is none of the reagent(s) left at the conjugate zone.

As the concentration of the targeting agent and/or the labels increasesat the capture zone, there may be an excess amount of the targetingagent and/or the labels for optimal formation of complex at the capturezone. In contrast, when the concentration of the targeting agent and/orthe labels is lower, there may be insufficient amounts of the targetingagent and/or the labels at the capture zone. Consequently, the totalamount of complex captured at the capture zone may be less than thetotal amount captured when the contact end of the chromatographic stripis contacted with test solution containing an optimal concentration ofthe targeting agent and/or the labels. The sensitivity of detection may,therefore be reduced.

Thus, it may be preferred that the targeting agent and/or labels are notreleasably immobilised to a conjugate zone of the chromatographic strip.

There is also provided according to the invention use of a kit of theinvention to test for the presence of an analyte in a test solution.

There is also provided according to the invention a labelled targetingagent for testing for the presence of an analyte in a test solution, thelabelled targeting agent being capable of binding the analyte or aderivative thereof but not being bound to the analyte or derivative,wherein the labelled targeting agent is provided with a plurality ofligands, each ligand capable of being bound by a label to allowdetection of the labelled targeting agent utilising the labels. Theinvention further provides use of a labelled targeting agent of theinvention to test for the presence of an analyte in a test solution.

Embodiments of the invention are illustrated with reference to FIG. 1.In the following discussion, the primary antibody corresponds to thetargeting agent, the haptens correspond to the ligands of the targetingagent, the labelled secondary antibody corresponds to the labellingagent, and the capture antibody corresponds to the capture moiety.

The major disadvantage of any membrane-based rapid assay is its reducedsensitivity compared with that of enzyme-linked immunoassay (EIA). Thisreduced sensitivity is due to the fact that, in a rapid assay,antigen-antibody reaction must reach completion within 15 to 20 minuteswithout the benefit of alternate washing and incubation steps. Inaddition, the antigen-antibody complex is detected by visual readout,without the help of signal amplification by enzymatic reactions. Thecombination of the reduction in sensitivity inherent in the rapid assayformat with that due to limited sample volume must be compensated for inan improved test system.

It is intended to amplify the detection signal by chemically couplingmultiple copies (N) of a hapten, such as biotin or fluorescein, to theprimary antibodies, which will be targeted to an analyte to be tested.This, together with labelling of the secondary antibodies (anti-hapten)with a label such as coloured particles (for example colloidal gold),will increase the detection signal.

FIG. 1 shows a comparison of assay sensitivity between a direct assaywithout amplification (prior art) and an indirect detection system withamplification according to the invention.

In FIG. 1 the analyte to be tested for is CT-lipopolysaccharide(CT-LPS). In the prior art assay without amplification (directdetection, without signal enhancement according to the invention), alabelled antibody is targeted to a specific antigen on the analyte. InFIG. 1A this is shown as an anti-LPS antibody attached to colloidalgold. The antibody can react with the specific antigen (e.g. LPS) on ananalyte (e.g. CT-LPS) in a liquid sample and bind it. In order to detectthe analyte when bound to the labelled antibody, an immobilised captureantibody is used. This is shown in FIG. 1A also as an anti-LPS antibodywhich recognises a different specific antigen on the same analyte.

The CT-LPS analyte (shown as reference 1 in FIG. 1A) is multimeric. Itis extracted from CT bacteria, for example by detergent or heattreatment. The capture antibody and the labelled antibody bind to adifferent part of the CT-LPS multimer.

According to the invention, the primary antibody specific for theanalyte is not labelled directly (indirect detection). The primaryantibody is provided with multiple copies (preferably 3-9) of a hapten,particularly biotin. The multiple haptens on the primary antibody can betargeted by anti-hapten labelled secondary antibodies. In the example ofFIG. 1B these are anti-biotin antibodies with colloidal gold labels.Multiple secondary antibodies can bind to a single primary antibody,leading to amplification of the number of labels per analyte. Theconjugate of the analyte and the primary antibody (labelled withsecondary antibodies) can then be captured and detected in theconventional manner using a capture antibody.

It is to be expected that detection of analyte according to theseembodiments of the invention would normally be carried out by firstreacting the primary antibody with a sample to be tested (which may ormay not contain the analyte). Once primary antibody has reacted with anyanalyte present, it can then subsequently be labelled with the secondaryantibody. The conjugate of any analyte with the primary antibody andsecondary antibody labels can then be captured by a capture antibody.

However, it is not absolutely essential to the underlying principle ofthe invention that the antibody-antigen and antibody-hapten reactions becarried out in exactly this order. Those of skill in the art willappreciate that under certain circumstances it might be possible or evenpreferable to react a secondary labelled antibody with the primaryunlabelled analyte-targeting antibody as a first step, followed byreaction of the conjugate with any analyte in a test sample andsubsequent capture of the labelled analyte.

It is also not excluded that there might be the possibility of firstlycapturing any analyte on the capture antibody and subsequently carryingout the primary and secondary antibody binding reactions (in whicheverorder is appropriate), as long as the primary and secondary antibodiesare bound to each other before reaching the capture zone.

It will be appreciated that in certain circumstances it may be anadvantage to have a first reaction step in which the anti-analyteantibody (or targeting agent) has optimal conditions (time, temperature,reagents etc) in which to react with any analyte present in a sample.This may particularly be the case where the analyte is in lowconcentration in a sample or is in some way difficult to access by theprimary antibody.

In such circumstances the best possible binding conditions for theprimary antibody may be found giving the best possibility of detectinganalyte in a sample. Thus, it will be appreciated that there may beconditions which will favour binding of an unlabelled primary antibodyto an analyte over the binding of a labelled primary antibody to ananalyte (as would be the case in the prior art direct labelling method).

Furthermore, since the avidity of the secondary antibody (or labellingagent) carrying the label for the hapten on the primary antibody(according to the invention) may be very high, it is another potentialadvantage of the invention that it is feasible to use relatively lowconcentrations of labelled antibody whilst yet obtaining relatively highlevels of (amplified) label attachment (via the primaryantibody/secondary antibody conjugate attachment to the analyte).

We have also recognised that in the prior art, direct labelling method,the label (e.g. the coloured particle: colloidal gold) may be attachedvia the antibody in quite close proximity to the analyte target (i.e.the LPS). This is shown schematically in FIG. 1A. For example aseparation between the analyte and the gold particle of about 12 nm is apossibility. In contrast, when using a primary antibody of the typeshown in FIG. 1, with multiple haptens and using anti-hapten labelledsecondary antibodies, it is possible to increase the separation betweenthe analyte and the label. For example a separation of about 24.2 nm ispossible.

Thus, in this invention, in the embodiment shown in FIG. 1B, it is mostlikely that the signal amplification (i.e. signal enhancement) is due tonot only more coloured particles captured by the capture antibody, butalso reduced steric hindrance encountered when gold-labelled antibodybinds to the epitope of specific antigen. In other words, increasing thespace between coloured particles and antigenic sites will reduce thesteric hindrance and allow more coloured particles to accumulate at thecapture zone.

A further possible reason for the enhanced sensitivity of detectionachieved with methods and kits of the invention may arise from anincreased probability of binding of label to the targeting agent due tothe presence of the plurality of ligands of the targeting agent.

In other embodiments of the invention, the analyte may be an antibody,for example an antibody raised by an individual against an antigen of aninfecting micro-organism such as HIV. In such embodiments, the primaryantibody (again provided with multiple copies of a hapten, such asbiotin or fluorescein) is targeted to the analyte antibody. As in otherembodiments described, the multiple haptens on the primary antibody canbe targeted by labelled anti-hapten secondary antibodies, therebyallowing amplification of the number of labels per analyte. However, inorder to capture a conjugate of the analyte and the primary antibody(labelled with secondary antibodies), an immobilised capture antigen isused which can be targeted by the analyte antibody.

Where the analyte is a human antibody, the primary antibody may be ananti-human Fc antibody or an anti-human antibody, e.g. IgG, IgM, or afragment thereof.

Further preferred embodiments of the invention are shown in FIG. 5.

It should be noted that the embodiments of the invention illustrated inFIGS. 1 and 5 are schematic. In reality, the labels may be much largerin relation to the other components than is shown and each label mayhave several moieties associated with it.

Further embodiments of the invention are now described by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1 shows schematically a comparison of conventional analytedetection with analyte detection according to an embodiment of theinvention;

FIG. 2 shows schematically a chromatographic strip used for carrying outembodiments of methods of the invention;

FIG. 3 shows results obtained by testing for Chlamydia trachomatis usinga conventional method (direct detection) and a method of the invention(indirect detection);

FIG. 4 shows results obtained by testing for HBsAg using a conventionalmethod (direct detection) and a method of the invention (indirectdetection);

FIG. 5 shows examples of embodiments of the invention;

FIG. 6 shows examples of derivatizing moieties captured with the analyteby a capture moiety (note that binding of analyte by labelled targetingagent is not shown);

FIG. 7 shows a comparison of the sensitivity of analyte detection usingan indirect detection method with that for direct detection, and theeffect on indirect detection of varying the number of ligands peranti-analyte antibody;

FIG. 8 shows the effect on the sensitivity of analyte detection byvarying the number of ligands per anti-analyte antibody when a fast flowrate dipstick membrane is used;

FIG. 9 shows a comparison of the sensitivity of analyte detection forone-step and two-step detection assays;

FIG. 10 shows the results of analyte detection using a FITC coupled toanti-analyte antibody; and

FIG. 11 shows a comparison of HBsAg detection using a method of theinvention with a commercially available detection system.

In the following examples, direct detection refers to analyte detectionusing a prior art method of analyte detection similar to that describedwith reference to FIG. 1A. Indirect detection refers to analytedetection using a method of the invention. Details of the method usedare given in each example.

EXAMPLE 1 Signal Enhancement Using an Anti-Lipo-Polysaccharide (LPS)Antibody Coupled to Biotin in a Dipstick Immunoassay to Test forChlamydia trachomatis (CT) Aim

To investigate the sensitivity of detection of CT using an anti-LPSmonoclonal antibody coupled to biotin (as a targeting agent) and acolloidal gold labelled anti-biotin monoclonal antibody (as a labellingagent). Detection is carried out using a method of the second aspect ofthe invention in which the targeting agent and the labelling agent arereleasably immobilised to the chromatographic strip.

Experiment Set-Up Dipstick Design

See FIG. 2 which shows a schematic drawing of a chromatographic stripused to perform a method of the invention.

FIG. 2 shows:

A. Conjugate pad: B. Read-out zone (membrane); C. Absorbent pad; D.Primary antibody=anti-CT-LPS antibody coupled to biotin (targetingagent); E. Secondary antibody=anti-biotin-gold conjugate (labellingagent); F. Specific antibody capture zone; G. Capture zone for controlantibody. Membrane may be overlapped between A and B as well as B and C.Sample flow is shown by the bold arrow.

Capture zone, F: a monoclonal antibody (the capture moiety) against agenus-specific CT-LPS epitope is immobilised on the membrane.

Detection reagents (the targeting agent and the labelling agent) forindirect detection of CT according to a method of the invention: ananti-CT-LPS monoclonal antibody coupled to biotin (targeting agent) andan anti-biotin monoclonal antibody coupled to colloidal gold (labellingagent) are deposited onto zones D and E of the conjugate pad.

Detection reagents for direct detection of CT using a conventionalmethod: a colloidal gold labelled anti-CT-LPS monoclonal antibody.

Sample Preparation: Analyte to be Tested:

Men's urine: centrifuge 2 ml of urine, discard the supernatant andresuspend the pellet with sample buffer and heat for 15 min at 100° C.

Sample Buffer:

Standard sample buffer comprising salt, detergent and a blocker (such asBSA or powdered milk).

Sample running: sample extract (100 μl) is added to the conjugate pad(A), or the conjugate pad is immersed in a sample extract. The detectionreagents (D&E) are solubilised and begin to move with the sample flow bycapillary action along the strip. The molecules of CT-LPS (analyte)present in the sample are bound by the primary antibody (anti-CT-LPSantibody coupled to biotin). Secondary antibody (anti-biotin-gold)conjugates to the biotin on the primary antibody. As the sample passesinto the read-out zone (B) and passes over the zone (the capture zone)on which the capture antibody (F) has been immobilised, the complex istrapped. Colour develops in zone F in proportion to the amount of CT-LPSpresent in the sample.

Procedure control: as a control for direct detection, an anti-mouse-IgGantibody was immobilised at zone G. For indirect detection, ananti-biotin antibody was immobilised at zone G as a control.

Result

See FIG. 3 and Table 1 which shows a comparison of direct and indirectdetection as described above. The effect of the molecular ratio ofbiotin per antibody on assay sensitivity is also shown in Table 1 and inFIG. 7.

TABLE 1 Comparison of direct and indirect detection, and effect ofmolecular ratio of biotin per antibody on assay sensitivity (using AE99membrane, a slower flow rate membrane) Indirect detection (number ofbiotins per CT-LPS Direct anti-LPS Ab) (μl)* detection 2 4 6 8 9 10 1112 0 0 0 0 0 0 0 0 0 0 0.001 0 0 0 0.5 1 1 0.5 0.5 0.5 0.003 0 0 0 1 2 21.5 1.5 1.5 0.01 0.5 0.5 0.5 2 2.5 2.5 2 2 2 0.03 1 1 2.5 3 4 4 3.5 3.53.5 0.1 2 2.5 3 4 4.5 4.5 4 4 4 *1 μl contains 4.218 ng of LPS. Thedetection signal is on a scale of 1 to 5 with 5 being the strongest.

The results of Table 1 show:

(1) That the amplified detection assay of the invention can detectCT-LPS at a lower concentration than can be detected using the directdetection assay;(2) The detected signal was amplified (i.e. enhanced) using the assaymethod of the invention.

EXAMPLE 2 Assay for HBsAg Detection

Rapid tests are used in some developing countries to screen blood forHBsAg, particularly in blood banks that screen too few samples tojustify microtiter plate enzyme immunoassay (EIA) or the considerableexpense for the necessary equipment. The most commonly used rapid testsfor the detection of HBsAg in developing countries are agglutination,dipsticks and spot tests. Agglutination assays are insensitive,relatively non-specific, subjective and labour intensive. Spot testsrequire multiple reagents and are unsuitable for medium size throughputand pre-donation screening. In addition, these assays are considerablymore expensive than standard EIAs. Dipstick tests are also commerciallyavailable, and some of them have high levels of performance. Compared toEIA, the limit of sensitivity of these rapid tests is an order ofmagnitude lower and results in a lower percentage of HBV infectiousblood units detected. EIA itself is 3% less sensitive than genomicamplification (Table 2).

TABLE 2 Detection of HBV infectious blood units by different screeningassays Limit of % of HBV infectious blood Assay Detection unitsdetected* Agglutination 30 ng/ml 54 Dipstick 5 ng/ml 77 EIA 0.5 ng/ml 97Genomic 40 IU/ml 100 DNA *100% correspond to HBsAg detection by EIA plusHBV DNA detection by PCR in EIA negative blood units

Of the available rapid dipstick tests for HBsAg on the market, theAbbott Determine test seems to be the most sensitive (Table 3), with adetection limit of approximately 5 ng/ml, whereas the other tests rangefrom 12-25 ng/ml.

TABLE 3 Comparison of three commercial HBsAg rapid tests HBsAg (ng/ml)Company name 25 12 6 3 Abbott 3 2 1.5 0.5 Veda Lab 2 1 0 0 AlfaScientific 1 0 0 0 Signals are on a scale of 1 to 5 with 5 being thestrongest signal

Aim

To investigate the sensitivity of detection of HBsAg using an anti-HBsAgmonoclonal antibody coupled to biotin (targeting agent) and a colloidalgold labelled anti-biotin monoclonal antibody (labelling agent).Detection was carried out using a method of the second aspect of theinvention in which the targeting agent and the labelling agent arereleasably immobilised to the chromatographic strip.

Experiment Set-Up Dipstick Design: See FIG. 2

For capture line: a polyclonal antibody directed against agenus-specific HBsAg epitope (the ‘a’ epitope common to all HbsAgsubtypes) is immobilised on the membrane (capture moiety).

For Detection:

Direct: a colloidal gold labelled anti-HBsAg monoclonal antibody wasused for direct detection using a conventional method.Indirect: an anti-HBsAg monoclonal antibody coupled to biotin (targetingagent) as well as a colloidal gold labelled anti-biotin monoclonalantibody (labelling agent) are used.Sample: 50 μl of serum plus 10 μl Tween 20 at 2%.Running sample: add sample (60 μl) to a container and dip a dipstickinto the solution.Procedure control: an anti-mouse-IgG antibody is immobilised forprocedure control.

Result See FIG. 4 and Table 4.

TABLE 4 Comparison of direct (prior art) and indirect detection (signalenhancement according to the invention) using Purabind A-XP membrane (afaster flow rate membrane) Indirect detection (number of HBsAg Directbiotins/anti-HBsAg Ab) ng/ml detection 2 4 8 9 10 11 0 0 0 0 0 0 0 00.195 0 0 0 0.5 0.5 0 0 0.39 0 0 0 1 1 0.5 0.5 0.78 0 0 0.5 1.5 1.5 1 11.56 0.5 0.5 1 2.5 2.5 2 2 3.12 1 1 1.5 3.5 3.5 3 3 6.25 1.5 2 2 4.5 4.54 4 12.5 2 3 3 5 5 4.5 4.5 The detection signal is on a scale of 1 to 5with 5 being the strongest.

Detection using an indirect detection method of the invention alloweddetection of analyte at lower concentrations than by a conventionaldirect detection method.

For those analyte concentrations at which both methods (direct andindirect) were able to detect analyte (≧1.56 ng/ml), a stronger signalwas obtained using indirect detection compared to direct detection.

For the indirect detection method, the optimum number of biotinmolecules per molecule of antibody was 8 or 9.

Although one extra antibody is used in indirect detection compared todirect detection, the indirect detection method is still a one-stepsystem.

The results of Table 4 also show that indirect detection according tothe invention is more sensitive than commercially available rapid tests.To confirm this, the sensitivity of HBsAg detection using a method ofthe invention was directly compared with the sensitivity of detectionusing the commercially available Abbott Determine test. The results areshown in Table 5 and FIG. 11.

TABLE 5 Comparison of HBsAg detection between a method of the inventionand a commercially available Abbott rapid test Dipstick HBsAg (ng/ml)test 12 6 3 1.5 0.75 0.38 0.18 0 Indirect 5 4 3 2 1.5 1 0.5 0 Direct 21.5 0.5 0 0 0 0 0 (Abbott) Signals are on a scale of 1 to 5 with 5 beingthe strongest signal

Discussion and Conclusion from Examples 1 and 2

Compared with the prior art direct detection system, the detectionsignal was amplified (i.e. enhanced) by using an antibody coupled tobiotin and a gold-labelled anti-biotin antibody.

Compared to the sensitivity of HBsAg detection using presently availablerapid dipstick tests (approximately 5 ng/ml for the Abbott Determinetest, and 12-25 ng/ml for the other tests), tests of the invention areconsiderably more sensitive, allowing detection of as little as 0.38ng/ml.

The degree of sensitivity improvement depends on the molecular ratio ofbiotin per anti-LPS or HBsAg antibody.

The signal was enhanced by increasing the number of molecules of biotinper antibody from 4 to 8 or 9. When an anti-LPS or HBsAg monoclonalantibody was labelled with 8 or 9 biotins, the assay sensitivityincreased 30 fold for CT-LPS and 8 fold for HBsAg. The same strengthsignal (1) is obtained for 0.00111 CT-LPS using the assay of theinvention compared to 0.03 μl CT-LPS using the direct detection priorart assay. The 30-fold increase is particularly surprising because themaximum improvement in the sensitivity of analyte detection is expectedto be 8- or 9-fold where 8 or 9 biotins per antibody are used.

Two possible explanations for the increased accumulation of colouredparticles using a method of the invention are given below:

(1) The higher the ratio of biotin on antigen specific antibody, themore gold particles captured. However, beyond a certain number ofmolecules of biotin per antibody, the availability of biotin will notincrease. On the contrary, if too many biotins are provided, thereactivity of the antibody with antigen may reduce.(2) The indirect detection with coloured particles will increase thedistance between gold particles and epitope and thus reduce sterichindrance of their binding.

The sensitivity of analyte detection using an indirect method of theinvention was reduced when gold-labelled streptavidin was used comparedto indirect detection using gold-labelled anti-biotin antibody. Thisindicates that the sensitivity of detection may be reduced if thedistance between the labels and the capture moiety is too little or ifthe flexibility of the targeting and/or labelling agent is notsufficient.

Two layers of antibody conjugated to coloured particles will furtherincrease the distance between coloured particles and epitope.

The size of the coloured particles used for labelled anti-haptenantibody should be optimised to achieve maximum signal. For example, 30nm colloidal gold is used for CT-LPS assay and for HBsAg assay.

Other ligands could be used, such as FITC (see example 8).

The experiments described in examples 1 and 2 have been repeated using adifferent indirect method of the invention. Instead of immobilising thebiotinylated anti-analyte antibody and anti-biotin gold conjugate to thechromatographic strip, the reagents were mixed with the test solutioncontaining CT-LPS or HBsAg analyte before contacting the mixed solutionwith the contact end of the chromatographic strip. The results obtainedwere similar to those obtained for examples 1 and 2. This shows that theimproved sensitivity of analyte detection obtained using a method of theinvention does not depend on whether the reagents used are immobilisedto the chromatographic strip or initially present in the test solution.

EXAMPLE 3 Optimum Number of Ligands Per Targeting Agent for Membranes ofDifferent Flow Rates

We have found that the optimum number of biotins per anti-analyteantibody depends on the flow rate of the dipstick membrane. For slowerflow rate membranes (for example Schleicher & Schuell AE99 membrane,pore size 8 μm) optimum results are obtained with about 6-12 ligands pertargeting agent, more preferably 8-12 ligands, even more preferably 8-10ligands, and most preferably 8 or 9 ligands.

Table 6 and FIG. 8 show the effect of the number of biotins peranti-analyte antibody on the sensitivity of analyte detection where afaster flow rate membrane (Whatman Purabind A-RP membrane) is used.Indirect detection according to the invention was carried out by mixingbuffer solution containing CT-LPS analyte with biotinylated anti-CT-LPSantibody and anti-biotin gold conjugate and then contacting the mixedsolution with the contact end of the chromatographic strip.

TABLE 6 Optimum number of ligands per targeting agent for membranes ofdifferent flow rates Indirect detection (number of CT-LPSbiotins/anti-LPS Ab) (μl)* 6 9 12 14 18 20 0 0 0 0 0 0 0 0 0 0 0 0.5 0.50 0.003 0 0.5 0.75 1 1 0.75 0.01 0.5 1 1.5 2 2 1.5 0.03 1 2 2.5 3.5 3.52.5 0.1 2 3 3.5 4.5 4.5 4 *1 μl contains 4.218 ng of LPS Signals are ona scale of 1 to 5 with 5 being the strongest signal.

To obtain a signal strength of 1 using an indirect detection method ofthe invention when only 0.003 μl of CT-LPS are present, the biotinylatedantibody should contain 14-18 biotins. Thus, the number of biotins perbiotinylated anti-CT-LPS antibody should be optimized for the flow rateof the membrane used.

EXAMPLE 4 Comparison of One-STEP and Two-Step Methods

In this example, the sensitivity of detection of CT-LPS analyte using aone-step assay (in accordance with an embodiment of the first aspect ofthe invention) and a two-step assay (not according to the invention)were compared.

To perform the one-step and two-step assays, a dipstick (Purabind A-RpMembrane, a Fast Flow Rate Membrane) comprising a contact end and anantibody (the capture antibody) immobilised to a capture zone of thedipstick remote from the contact end was used. The capture antibody iscapable of binding to the CT-LPS analyte (an anti-CT-LPS antibody). Abiotinylated anti-CT-LPS antibody comprising 14 biotins per antibody(the targeting agent) and anti-biotin antibody labelled with colloidalgold (the labelling agent) were used to detect the CT-LPS analyte.

The one-step assay was carried out by mixing a test solution containingCT-LPS with biotinylated anti-CT-LPS antibody andanti-biotin-antibody-gold conjugate, then contacting the mixed solutionwith the contact end of the dipstick, allowing the solution to reach thecapture zone by capillary action, and detecting for the presence of goldlabel at the capture zone. Thus, the analyte, targeting agent andlabelling agent are wicked up the dipstick simultaneously in a singlestep.

The two-step assay was carried out by mixing 50 μl buffer solution withCT-LPS and biotinylated anti-CT-LPS antibody, then contacting the mixedsolution with the contact end of the dipstick, and allowing the solutionto reach the capture zone by capillary action. Then, the contact end ofthe dipstick was contacted with 100 μl of a suspension of theanti-biotin-gold conjugate, and this was allowed to travel to thecapture zone by capillary action. Then, the presence of gold label wasdetected for at the capture zone. Thus, the targeting agent andlabelling agent are wicked up the dipstick separately in two distinctsteps.

The results of the comparison of the one-step and two-step assays areshown in table 7 and FIG. 9.

TABLE 7 Comparison of one-step and two-step assays CT-LPS (μl)* One-stepTwo-step 0 0 0 0.001 0.5 0 0.003 1 0 0.01 2 0.5 0.03 3 1 0.1 4.5 2 *1 μlcontains 4.218 ng of LPS Signals are on a scale of 1 to 5 with 5 beingthe strongest signal.

The results of Table 7 and FIG. 9 show that the sensitivity of CT-LPSdetection using the one-step CT-LPS assay is 10 times greater than thetwo-step CT-LPS assay (the same strength signal (1) is obtained for0.003 μl of CT-LPS using the one-step assay as with 0.03 μl CT-LPS usingthe two-step assay).

We have also found that the quality and visibility of the signal lineproduced at the capture zone of the dipstick is greater for the one-stepassay than the two-step assay.

EXAMPLE 8 Use of FITC as Targeting Agent Ligand

In this example the sensitivity of analyte detection using direct andindirect detection methods was again compared. However, here the ligandused in the indirect detection method was fluorescein isothiocyanate(FITC) rather than biotin. Direct (prior art) detection was carried outusing a similar method to that described in Example 1. Indirectdetection according to the invention was carried out by mixing buffersolution containing CT-LPS analyte with FITC coupled to anti-CT-LPSantibody and anti-FITC antibody labelled with colloidal gold. Then themixed solution was contacted with the contact end of the chromatographicstrip. The number of FITC ligands per anti-analyte antibody was varied.The results obtained are shown in Table 8 and FIG. 10, and are verysimilar to those obtained for Example 1. The optimum number of FITCmolecules per anti-CT-LPS antibody is about 7-11.

TABLE 8 Use of FITC as a targeting agent ligand (AE99 membrane) Indirectdetection CT-LPS Direct (number of FITCs/anti-LPS Ab) (μl)* detection 67 9 11 0 0 0 0 0 0 0.001 0 0 1 1 1 0.003 0 0.5 2 2 2 0.01 0.5 1 2.5 2.52.5 0.03 1 2 3.5 4 3 0.1 2 3 4.5 4.5 4 *1 μl contains 4.218 ng of LPSSignals are on a scale of 1 to 5 with 5 being the strongest signal.

These results demonstrate that the improved sensitivity of analytedetection obtained using an indirect method of the invention is notrestricted to the use of biotin.

Figure Legends FIG. 1

A) Schematic representation of conventional detection of analyte (directdetection). The figure shows a CT bacterium (1) bound by an anti-LPSantibody (2) immobilised to a solid phase (3) and an anti-LPS antibody(4) labelled with colloidal gold (5) bound to the CT bacterium (1).B) Schematic representation of detection of analyte according to anembodiment of the invention. The FIG. 10 shows a CT bacterium (6) boundby an anti-LPS antibody (7) immobilised to a solid phase (8) and ananti-LPS antibody (9) coupled to 8 biotins (10) [(9) and (10) form thetargeting agent]. Each biotin is bound by an anti-biotin antibody (11)labelled with colloidal gold (12) [(11) and is (12) form the labellingagents]

FIG. 5

Different embodiments of the invention are shown schematically:

A) Analyte (20) captured by a capture moiety (21) immobilised to a solidphase (22). A primary moiety (23) [for example an anti-analyte mouseantibody] of the targeting agent is bound to the analyte (20) and isitself bound by a secondary moiety (24) [for example an anti-mouseantibody] of the targeting agent. The secondary moiety (24) comprises aplurality of ligands (25). The ligands are bound by labelling agents(26) comprising labels (27) [for example anti-ligand antibodiesconjugated with coloured particles].B) Analyte (30) captured by a capture moiety (31) immobilised to a solidphase (32). A targeting agent (33) [for example an anti-analyteantibody] provided with a plurality of ligands (34) is bound to theanalyte (30) and is itself bound by a plurality of primary moieties (35)[for example anti-ligand mouse antibodies] of the labelling agent. Eachprimary moiety (35) is bound by a secondary moiety (36) of the labellingagent comprising a label (37) [for example an anti-mouse antibodyconjugated with a coloured particle].C) Analyte (40) captured by a capture moiety (41) immobilised to a solidphase (42). A targeting agent (43) provided with a plurality of ligands(44) is bound to the analyte (40) and is itself bound by a plurality oflabelled primary moieties (45) of the labelling agent. Each labelledprimary moiety (45) is bound by a labelled secondary moiety (46) of thelabelling agent. The labels are shown as (47).

FIG. 6

Further embodiments of the invention are shown schematically:

A) Analyte (110) bound to a derivatizing moiety (112) provided with aplurality of ligands (114) captured by a capture moiety (116)immobilised to a capture zone of the chromatographic strip (118).B) Analyte (120) bound to a first derivatizing moiety (122) providedwith a plurality of ligands (124). A second derivatizing moiety (126) isbound to a ligand of the first derivatizing moiety. The secondderivatizing moiety is captured by a capture moiety (128) immobilised toa capture zone of the chromatographic strip (130).C) Analyte (140) bound to a first derivatizing moiety (142). A secondderivatizing moiety (144) bound to the first derivatizing moiety isprovided with a plurality of capture ligands (146). A capture moiety(148) immobilised to a capture zone of chromatographic strip (150) bindsto one of the capture ligands.

1. A method for testing for the presence of an analyte in a testsolution which comprises the following steps: a) providing achromatographic strip having a contact end for contacting the testsolution and a capture moiety immobilised at a capture zone of thechromatographic strip, the capture moiety comprising a member of aligand/anti-ligand binding pair capable (other than by nucleic acid basepairing interaction) of binding the analyte, or a derivative thereof, asthe other member of the ligand/anti-ligand binding pair; b) contactingthe contact end of the chromatographic strip with the test solution toallow analyte, or derivative thereof, to travel to the capture zone bycapillary action and be captured by the capture moiety; c) contacting alabelled targeting agent with the capture zone, the labelled targetingagent being capable of binding the analyte, or a derivative thereof,thereby allowing labelled targeting agent to be captured at the capturezone as part of a complex comprising the capture moiety, the analyte orderivative and the labelled targeting agent, wherein the labelledtargeting agent comprises a plurality of ligands and each of two or moreligands are bound to a label; and d) detecting for the presence of labelat the capture zone.
 2. A method for testing for the presence of ananalyte in a test solution which comprises the following steps: a)providing a chromatographic strip having a contact end for contactingthe test solution and a capture moiety immobilised at a capture zone ofthe chromatographic strip remote from the contact end, the capturemoiety comprising a member of a ligand/anti-ligand binding pair capableof binding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair; b) contacting a targeting agent,capable of binding the analyte or derivative thereof, with the testsolution to allow binding of the targeting agent to analyte orderivative in the test solution, the targeting agent being provided witha plurality of ligands; c) binding a label to each of two or moreligands of the targeting agent; d) contacting the contact end of thechromatographic strip with the test solution to allow analyte, orderivative thereof, bound to the labelled targeting agent to travel tothe capture zone by capillary action and be captured by the capturemoiety; and e) detecting for the presence of label at the capture zone.3. A method for testing for the presence of an analyte in a testsolution which comprises the following steps: a) providing achromatographic strip having: i) a contact end for contacting the testsolution; ii) a capture moiety immobilised at a capture zone of thechromatographic strip remote from the contact end, the capture moietycomprising a member of a ligand/anti-ligand binding pair capable ofbinding (other than by nucleic acid base pairing interaction) theanalyte, or a derivative thereof, as the other member of theligand/anti-ligand binding pair; and iii) a targeting agent releasablyimmobilised at a conjugate zone of the chromatographic strip between thecontact end and the capture zone, the targeting agent being providedwith a plurality of ligands and being capable of binding the analyte orderivative thereof; b) contacting a plurality of labels with the testsolution; c) contacting the contact end of the chromatographic stripwith the test solution to allow test solution to travel through theconjugate zone to the capture zone, thereby releasing targeting agentfrom the conjugate zone so that each of two or more ligands of thereleased targeting agent are bound by a label and so that releasedtargeting agent bound to the labels can travel with analyte orderivative in the test solution to the capture zone and be captured atthe capture zone as part of a complex formed between the capture moiety,analyte or derivative, the targeting agent, and the labels; and d)detecting for the presence of label at the capture zone.
 4. A methodaccording to claim 3 in which the labels are releasably immobilised tothe conjugate zone of the chromatographic strip so that the labels arecontacted with is the test solution as it travels to the capture zone,thereby releasing the labels to allow them to bind to the releasedtargeting agent.
 5. A method according to claim 3 in which the labelsare releasably immobilised to the chromatographic strip instead of thetargeting agent, and the targeting agent is contacted with the testsolution in step (b) instead of the labels.
 6. A method for testing forthe presence of an analyte in a test solution which comprises thefollowing steps: a) providing a chromatographic strip having a contactend for contacting the test solution and a capture moiety immobilised ata capture zone of the chromatographic strip remote from the contact end,the capture moiety comprising a member of a ligand/anti-ligand bindingpair capable of binding (other than by nucleic acid base pairinginteraction) the analyte, or a derivative thereof, as the other memberof the ligand/anti-ligand binding pair; b) contacting thechromatographic strip with the test solution to allow analyte, orderivative thereof, to travel to the capture zone by capillary actionand be captured by the capture moiety; c) either: (i) contacting atargeting agent capable of binding the analyte or derivative thereofwith the test solution, the targeting agent being provided with aplurality of ligands, binding a label to each of two or more ligands ofthe targeting agent, and contacting the contact end of thechromatographic strip with the test solution to allow labelled targetingagent in the test solution to travel by capillary action to the capturezone and bind to analyte or derivative captured at the capture zone; or(ii) contacting the contact end of the chromatographic strip with asolution containing a targeting agent capable is of binding the analyteor derivative thereof, the targeting agent being provided with aplurality of ligands and each of two or more ligands being bound by alabel, allowing labelled targeting agent in the solution to travel bycapillary action to the capture zone and bind to analyte or derivativecaptured at the capture zone; and d) detecting for the presence of labelat the capture zone.
 7. A method for testing for the presence of ananalyte in a test solution which comprises the following steps: a)providing a chromatographic strip having a contact end for contactingthe test solution and a capture moiety immobilised at a capture zone ofthe chromatographic strip, the capture moiety comprising a member of aligand/anti-ligand binding pair capable (other than by nucleic acid basepairing interaction) of binding the analyte, or a derivative thereof, asthe other member of the ligand/anti-ligand binding pair; b) contactingthe contact end of the chromatographic strip with the test solution toallow analyte, or derivative thereof, to travel to the capture zone bycapillary action and be captured by the capture moiety; c) contacting atargeting agent, capable of binding the analyte, or derivative thereof,with the capture zone to allow targeting agent to bind to analyte orderivative captured at the capture zone, the targeting agent beingprovided with a plurality of ligands, each of two or more ligands of thetargeting agent being bound to a label; and d) detecting for thepresence of label at the capture zone.
 8. A method according to claim 1,2, 6, or 7 in which binding of the labels to the ligands of thetargeting agent is carried out in the test solution.
 9. A methodaccording to claim 8 in which the labels are provided by labellingagents which are added separately to the test solution at substantiallythe same time as the is targeting agent is contacted with the testsolution.
 10. A method according to claim 8 in which the targeting agentis pre-incubated with the test solution before the labels are bound tothe targeting agent.
 11. A method according to any preceding claim inwhich a plurality of labels are bound to at least one of the ligands ofthe targeting agent.
 12. A kit for testing for the presence of ananalyte in a test solution which comprises: i) a chromatographic stripas defined in claim 2; ii) separately from the chromatographic strip, atargeting agent capable of binding the analyte or derivative thereof,the targeting agent being provided with a plurality of ligands, and aplurality of labelling agents each bound to a ligand of the targetingmoiety, each labelling agent being provided with a label.
 13. A kit fortesting for the presence of an analyte in a test solution whichcomprises: i) a chromatographic strip as defined in claim 3; and ii) aplurality of labelling agents capable of binding to a ligand of thetargeting moiety, each labelling agent being provided with a label. 14.A kit for testing for the presence of an analyte in a test solutionwhich comprises: i) a chromatographic strip as defined in claim 2 or 3;and a labelling agent releasably immobilised to a conjugate zone of thechromatographic strip between the contact end and the capture zone. 15.A kit according to claim 12 in which the labelled targeting agent is indry form.
 16. A kit according to claim 13 in which the labelling agentsare in dry form, or a kit according to claim 14, where the targetingagent is separate from the dipstick, in which the targeting agent is indry form.
 17. A kit according to any of claims 12 to 16 in which thetargeting agent comprises an antibody coupled to a plurality of ligands.18. A kit according to any of claims 12 to 16 in which the targetingagent comprises: a primary moiety capable of binding to the analyte orderivative; and a secondary moiety capable of binding to the primarymoiety, the secondary moiety being coupled to a plurality of ligands.19. A kit according to claim 18 wherein the primary and/or secondarymoiety of the targeting agent is an antibody.
 20. A kit according to anyof claims 12 to 19 in which each labelling agent comprises a labelledantibody.
 21. A kit according to any of claims 12 to 19 in which eachlabelling agent comprises: a primary moiety capable of binding to aligand of the targeting agent; and a labelled secondary moiety capableof binding to the primary moiety.
 22. A kit according to claim 21 inwhich the primary and/or secondary moiety of the labelling agent is anantibody.
 23. A kit according to claim 21 or 22 in which the primarymoiety of the labelling agent is labelled.
 24. A kit according to any ofclaims 12 to 23 in which the labels comprise visually detectable labels,preferably coloured particles, or luminescent labels, preferablyfluorescent labels.
 25. A kit according to any of claims 12 to 24 inwhich the labels comprise an enzyme capable of converting a substrateinto a coloured product, or a luminescent product, preferably afluorescent product.
 26. A kit according to any of claims 12 to 25 inwhich the analyte is a CT analyte, or an HBsAg.
 27. Use of a kitaccording to any of claims 12 to 26 to test for the presence of ananalyte in a test solution.
 28. A method according to any of claims 1 to11 in which the analyte is a CT analyte, or an HBsAg.
 29. A labelledtargeting agent for testing for the presence of an analyte in a testsolution, the labelled targeting agent being capable of binding theanalyte or a derivative thereof but not being bound to the analyte orderivative, wherein the labelled targeting agent is provided with aplurality of ligands, each ligand being bound by a label to allowdetection of the labelled targeting agent utilising the labels.
 30. Atargeting agent according to claim 29 provided with about 6-50 ligands.31. Use of a targeting agent according to claim 29 or 30 to test for thepresence of an analyte in a test solution.